Glutathione (GSH; g-glutamyl-cysteinyl-glycine) is the predominant thiol produced by aerobic eukaryotes and Gram-positive bacteria. It is believed to protect aerobic organisms from oxygen toxicity and to participate in a multitude of functions. GSH acts as a slowly autooxidizing reserve of cysteine and as a cofactor in the detoxification of peroxides, epoxides, and other products resulting from reaction with oxygen. It is a cofactor in the reduction of disulfides and ribonucleotides and in the isomerization of protein disulfides. Thiols are the most reactive nucleophiles in the cell at physiological pH, and when exposed to atmospheric oxygen are oxidized to disulfides (RSH/RSSR=10.sup.-16). Glutathione reductase (GSR; E.C.1.6.4.2) catalyzes the NADPH-dependent reduction of intracellular oxidized glutathione (GSSG) and thereby maintains a reducing environment in the cell (GSH/GSSG&gt;100). GSH was once thought to be ubiquitous. However, many organisms do not produce GSH but instead produce alternative thiols. Fahey et al. (1978) J. Bacteriol. 133:1126-1129; Fahey et al. (1991) in Meister (ed.) Advances in Enzymology and Related Areas of Molecular Biology 64:1-53 (John Wiley and Sons); Fairlamb (1989) Parisitol. 99S:93-112; Newton et al. (1989), in Vina (ed.), Glutathione: Metabolism and Physiological Functions pp. 69-77 (CRC Press, Boca Raton. Fla.); Newton et al. (1993) J. Bacteriol. 175:2734-2742; Sakuda et al. (1994) Biosci. Biotechnol. Biochem. 58:1347-1348; and Spies et al. (1994) Eur. J. Biochem. 224:203-213.
For example, Staphylococcus aureus produces Coenzyme A (CoA) as its major thiol instead of glutathione. Newton et al. (1996) J. Bacteriol., in press. CoA is slightly more stable than glutathione to heavy metal-catalyzed auto-oxidation and provides a stable redox buffer similar to that provided by GSH in other organisms. S. aureus maintains millimolar levels of reduced CoA as its predominant thiol and, like most of the Gram-positive bacteria, essentially no GSH. Newton et al. (1996), supra; Newton et al. (1989), supra. CoA is required throughout metabolism as a cofactor in acyl transfer reactions and likely has additional functions in S. aureus analogous to those of GSH in other organisms.
Other organisms that utilize alternative thiols produce an enzyme analogous to GSR. The preferred substrate for such an enzyme is the disulfide of the predominant thiol in the cell. Shames et al. (1986) Biochemistry 25:3519-3526; Swerdlow et al. (1983) J. Bacteriol. 153:475-484. All such enzymes belong to a widespread family of pyridine nucleotide dependent disulfide reductases that include GSR, lipoamide dehydrogenase, and mercuric reductase. Most of these enzymes are homodimeric flavoproteins of M.sub.r .about.100 kD that utilize a conserved active-site disulfide bond to effect catalysis.
Antimicrobial agents commonly used to combat microorganism infections generally interfere with a critical step in the metabolism of the microorganism resulting in growth inhibition or death thereof. However, pathogenic microorganisms, including staphylococci and enterococci, are developing resistance, and in many cases multiple resistances, to existing antimicrobial agents.
S. aureus is an opportunistic pathogen of increasing medical concern. It can be aggressively invasive, spreading rapidly through soft tissues, directly invading bones and even entering the bloodstream in which it may produce septic shock and disseminated intravascular coagulation. Two categories of disease may be ascribed to staphylococci: those related to toxins produced by the organism (S. aureus exclusively), including gastroenteritis, toxic shock syndrome, scalded skin syndrome, and the like; and those related to direct invasion and systemic spread of the organism, including dermal infections, bone and joint infections, staphylococcal pneumonia and empyema, meningitis, cerebritis, endocarditis, bacteremia, septic shock, and the like.
Strains of .beta.-lactam antibiotic resistant staphylococci (BLARS), otherwise referred to as methicillin-resistant S. aureus (MRSA), have become a widespread cause of fatal nosocomial infection. Infections caused by such resistant staphylococci are treated predominantly by the "last resort" antibiotic, vancomycin. Newer antimicrobial agents that may be effective against staphylococcal infections include the investigational agent teichoplanin and the quinolones; however, recent data indicate increasing quinolone resistance. Since vancomycin resistance would essentially exhaust the current antibiotic therapeutic arsenal, it is now mandatory to identify new cellular targets and new chemotherapeutic agents effective against MRSA.
Accordingly, there is a need for new antimicrobial agents to which microorganisms are susceptible. The ability to discover and use such agents would be augmented by the availability of new cellular targets. Acquired resistance that protects against or compensates for disruption of one metabolic pathway by a particular class of antimicrobial agents would be unlikely to have a similarly protective or compensatory effect for disruption of a distinct metabolic pathway.